title: Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells.
abstract: BACKGROUND
  Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.
  
  
  METHODS
  Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.
  
  
  RESULTS
  Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.
  
  
  CONCLUSIONS
  The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.
authors.affiliation: Hannover Clinical Trial Center GmbH, Carl-Neuberg-Strasse. 1/k27, 30625 Hannover, Germany. Electronic address: email.sz555@gmail.com.
authors.forename: Silke
authors.initials: S
authors.lastname: Zimmermann
authors.forename: Christiane
authors.initials: C
authors.lastname: Hollmann
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Stachelhaus
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology; D018955:CD36 Antigens / Q000276:immunology; D002478:Cells, Cultured; D041905:Erythroid Cells / Q000276:immunology*; D005312:Fetal Blood / Q000276:immunology*; D005690:Galactose / Q000276:immunology; D006021:Glycophorins / Q000276:immunology; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D011990:Receptors, Transferrin / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018955:CD36 Antigens; C068126:CD71 antigen; C409972:GYPA protein, human; D006021:Glycophorins; D011990:Receptors, Transferrin; D005690:Galactose
keywords: 4′-6-Diamidino-2-phenylindol; APAAP; APC; Alkaline phosphatase anti-alkaline phosphatase; Allophycocyanine; BFU—E; BSA; Bovine serum albumin; Burst-forming unit—erythoid; CD; Cluster of differentiation; DAPI; DMSO; DNA; DSMZ; Deoxyribonucleic acid; Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures); Dimethylsulfoxide; FACS; FC receptor; FCS; FISH; FITC; FSC; FcR; Fetal calf serum; Fetal nucleated red blood cells; Fluoresceinthiocyanate; Fluorescence in situ hybridization; Fluorescence-activated cell sorter; Forward scattered light; GPA; GalNAc; Glycophorin A; HRPO; Horseradish peroxidase; Immunophenotyping; MNCs; Monoclonal antibody; Mononuclear cells; NRBC; Nucleated red blood cell; PBS; PCR; PE; Phosphate-buffered saline; Phycoerythrin; Polymerase chain reaction; Prenatal diagnostics; RT; Room temperature; SBA; SSC; STR; Short tandem repeats; Side scattered light; Soybean agglutinin; TBS; Terminal α- or β-N-acetylgalactosamine; Tris; Tris(hydroxymethyl)aminomethane; Tris-buffered saline; mAb